Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, to molecules and surfaces is of considerable utility in biotechnology and medicine. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:HO—CH2CH2O—(CH2CH2O)n—CH2CH2—OH
The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol represents the following structural unit:—CH2CH2O—(CH2CH2O)n—CH2CH2—where n typically ranges from about 2 to about 4000.
PEG is commonly used as methoxy-PEG-OH, or MPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification. The structure of MPEG is given below.CH3O—(CH2CH2O)n—CH2CH2—OH
Random or block copolymers of ethylene oxide and propylene oxide, shown below, are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications.
 HO—CH2CHRO(CH2CHRO)nCH2CHR—OH
wherein each R is independently H or CH3.
PEG is a polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule “conjugate” soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
To couple PEG to a molecule, such as a protein, it is often necessary to “activate” the PEG by preparing a derivative of the PEG having a functional group at a terminus thereof. The functional group is chosen based on the type of available reactive group on the molecule that will be coupled to the PEG. For example, the functional group could be chosen to react with an amino group on a protein in order to form a PEG-protein conjugate.
The use of polypeptides, including proteins, for therapeutic applications has expanded in recent years mainly due to improved methods for recombinant expression of human polypeptides from various expression systems and the ability to deliver these polypeptides in vivo with improved properties. Many of the drawbacks associated with polypeptide therapeutics, including short circulating half-life, immunogenicity and proteolytic degradation, can and have been improved by methods such as gene therapy, epitope mutations by directed or shuffling mutagenesis, shielding of the epitope regions by natural or synthetic polymers, fusion proteins, and incorporation of the polypeptide into drug delivery vehicles for protection and slow release.
Polymer modification of proteins, such as covalent attachment of poly(ethylene glycol), has gained popularity as a method to improve the pharmacological and biological properties of therapeutically useful proteins. For example, poly(ethylene glycol) conjugated proteins are known to have significantly enhanced plasma half-life, reduced antigenicity and immunogenicity, increased solubility and decreased proteolytic degradation. Factors that affect the foregoing properties are numerous and include the number of poly(ethylene glycol) chains attached to the protein, the molecular weight and structure of poly(ethylene glycol) chains attached to the protein, the chemistries used to attach the poly(ethylene glycol) to the protein, and the location of the poly(ethylene glycol) sites on the protein.
A variety of methods have been developed to non-specifically attach poly(ethylene glycol) to proteins. Most commonly, electrophilically-activated poly(ethylene glycol) is reacted with nucleophilic side chains found on proteins. Attaching an activated poly(ethylene glycol) to the α-amine and ε-amine groups found on lysine residues and at the N-terminus result in conjugates consisting of a mixture of products as disclosed in U.S. Pat. No. 6,057,292. For example, the conjugate may consist of a population of conjugated proteins having varying numbers of poly(ethylene glycol) molecules attached per protein molecule (“PEGmers”), ranging from zero to the number of α- and ε-amine groups in the protein. For a protein molecule that has been singly modified, the poly(ethylene glycol) moiety may be attached at any one of a number of different amine sites. This type of non-specific PEGylation can result in partial or complete loss of the therapeutic utility of the conjugated protein.
Several methods for site-directed or selective attachment of PEG have been described. For example, a site-directed approach for conjugating poly(ethylene glycol) to the N-terminal α-amine of a protein is disclosed in WO 99/45026 and U.S. Pat. Nos. 5,824,784 and 5,985,265. WO 99/03887 and U.S. Pat. Nos. 5,206,344 and 5,766,897 relate to the site-directed PEGylation of cysteine residues that have been engineered into the amino acid sequence of proteins. While these methods offer some advantages over non-specific attachment, there is a continuing need for improved methods and reagents for providing site-specific polymer conjugated proteins.